Method of expressing genes in mammalian cells

ABSTRACT

The present invention relates to a method of expressing a heterologous gene in mammalian cells and a recombinant DNA construct for use in the method. The invention also relates to a method of specifically killing cells which constitutively express AFP.

This application is a divisional of application Ser. No. 08/148,058,filed Nov. 4, 1993.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method of expressing a heterologousgene in mammalian cells, and a recombinant DNA construct for use in themethod. More particularly the invention relates to a recombinant DNAobtained by linking a gene coding for a cancer-cell toxin to certain ofthe transcriptional regulatory regions of the human α-fetoprotein (AFP)gene so that the toxin gene is transcribed selectively in hepatic cancercells.

2. Description of the Related Art

Cancer of all forms is one of the major causes of morbidity throughoutthe world. Research in the area of cancer chemotherapy has produced avariety of antitumor agents which have differing degrees of efficacy. Avariety of cancer-therapeutic agents are known, for example, alkylatingagents, antimetabolites, alkaloids and carcinostatic antibiotics.Standard clinically used agents include adriamycin, actinomycin D,methotrexate, 5-fluorouracil, cis platinum, vincristine and vinblastine.However, these presently available antitumor agents are known to havevarious disadvantages such as toxicity to healthy cells and resistanceof certain tumor types.

Hepatocellular carcinoma (HCC) is one of the major malignant diseases inthe world today; the greatest incidence being in Japan, China, otherparts of the Asia and sub-Saharan Africa. Recent evidence suggests thatthe incidence of hepatocellular carcinoma in Europe and North America isincreasing. The disease is estimated to be responsible for or involvedin up to approximately 1,250,000 deaths a year and as such isnumerically one of the world's major malignant diseases.

The prognosis of HCC is poor with the world-wide frequency rate almostequalling the mortality rate. After diagnosis, the median survival timeis less than four months. Long-term survival, defined as survival longerthan one year after the diagnosis, is seen only occasionally. Most HCCpatients succumb to either the complications of liver failure with orwithout massive bleeding, or to the general effects of a large tumorburden, with cachexia, malnutrition, infection and sepsis. Thoughdistant metastases occur (up to 90% of patients have metastatic tumorsat the time of death), hepatic disease most often limits survival.Consequently, therapies directed towards the control of hepatic tumorsare appropriate, although it will be appreciated that treatment of themetastatic disease is also of great importance. (Berk. P. (Ed) Seminarsin Liver Disease 4, No. 2, Thieme-Stratton Inc. N.Y. N.Y. (1984).

Current therapies available to the clinician are on the wholeineffective as a cure for this disease (Nerenstone et al., CancerTreatment Review 15, 1-31 (1988). Systemic single and combination agentchemotherapy and radiation are relatively ineffective. Since a cancercell of a patient originates from a normal cell of the same patient,these known agents fail to clearly discriminate cancer cells from normalcells, thus causing various adverse effects. Therefore, use of the knowncancer-therapeutic agents is significantly limited in various ways.

To date, surgery continues to be the only potential cure. However, atthe time of diagnosis, the overwhelming majority of patients are notable to undergo radical surgery. In certain studies (Nerenstone et al.supra) it was found that less than 3% of patients were consideredcapable of undergoing surgery and of the small percentage that do,approximately 50% suffer from postoperative morbidity (Nerenstone et al.supra). However, it is appreciated by those skilled in the art thatnovel approaches and entities for cancer therapies are required.

Gene therapy involves the stable integration of new genes intoparticular target cells and the expression of those genes, once they arein place, to alter the phenotype of that particular target cell (forreview see Anderson, W. F. Science 226: 401-409, 1984; McCormick, D.Biotechnology 3: 690-693, 1985).

Genetic ablation takes advantage of tissue specificity of certain generegulatory elements to express a toxin gene in a cell specific manner.For example, Goring et al. (1987) Science 235: 456-458 have shown thatthe γ2-crystallin promoter is able to direct expression of a linked lacZgene in the lens fiber cells in transgenic mice in a developmentalmanner. When the diphtheria toxin A-chain (DT-A) gene is expressed in asimilar manner in the lens fiber cells in mice, microphthalmic micehaving lenses deficient of fiber cells are obtained (Brietman et al.,(1987) Science 238: 1563-1565). When an attenuated DT-A gene (tox 176)is used, transgenic mice which displayed predominately cataracts orclinical anophthalmia were obtained (Brietman et al. (1990) Mol. CellBiol. 10: 474-479).

Palmiter et al. (1987) Cell 50: 435-443 have used the elastase Ipromoter/enhancer to drive the expression of the DT-A gene in pancreaticacinar cells to yield mice lacking a normal pancreas.

Alternatively, the expression of a gene encoding an enzyme capable ofselective conversion of chemical agents to cytotoxic or cytostaticmetabolites has been proposed. (European Patent Application No. 0 415731). A molecular chimaera is constructed which consists of a naturaltranscriptional regulatory region attached to a gene encoding an enzymewhich is capable of converting a metabolite into the toxin. Uponadministration of the molecular chimaera to the patient and themetabolite, the enzyme is expressed in the cancer cells, which enzymeconverts the metabolite into the toxin thereby killing the cancer cells.However, this procedure requires the administration of two chemicals.Also, the possibility exists that the metabolite may be converted intothe toxin in the incorrect cells resulting in toxicity to normal cells.A method of controlling the expression of a toxin gene in cancer cellswould be useful.

SUMMARY OF THE INVENTION

The present invention is directed to a method of expressing aheterologous gene in mammalian cells and to a recombinant DNA constructfor use in the method.

Accordingly, one aspect of the present invention provides a method ofexpressing a heterologous gene in a mammalian cell comprising insertinga DNA construct into a mammalian cell, said construct comprising an AFPenhancer region and an AFP promotor functionally linked to theheterologous gene in the absence of an AFP silencer region, andexpressing the heterologous gene in the mammalian cells.

Another aspect of the present invention provides a method of killingcells which constitutively express AFP comprising inserting a DNAconstruct into a mammalian cell, said construct comprising an enhancerregion, an AFP silencer region and a promoter sequence functionallylinked to a direct toxin gene and expressing the toxin gene in themammalian cells under conditions such that only cells whichconstitutively express AFP are killed.

Also provided are mutated enhancer domains and silencer regions usefulin the methods of this invention.

Further advantages of the present invention will become apparent fromthe following description of the invention with reference to theattached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates CAT fusion genes containing the human albumin andα-fetoprotein (AFP) 5'-flanking regions. Positions of the enhancers (E₁(SEQ ID NO:1), E_(A) and E_(b) (nucleotides 1700 to 1712 of SEQ IDNO:12)), promoters (P) SEQ ID NOS:2, 3, 4 and 5),glucocorticoid--responsive element (G) and AT-rich elements are alsoindicated.

FIG. 1B illustrates CAT activities expressed in HuH-7 and huH-1/cl-2cells transfected with the CAT fusion genes shown in FIG. 1A. CATactivities as a percentage of pSV2-CAT activity are shown above theautoradiograms. Cm--chloramphenicol; 1-Ac, 1-acetate chloramphenicol;3-Ac, 3-acetate chloramphenicol.

FIG. 1C illustrates the effects of dexamethasone on CAT activities inHuH-7 and huH-1/cl-2 cells transfected with pAF1.0-CAT. Cm, 1-Ac and3-Ac are as defined in FIG. 1B. + and - indicate the presence andabsence of dexamethasone respectively.

FIG. 1D illustrates the AFP enhancer/SV40 early promoter fusion genes;Hi, Hind III; Bg, BglII.

FIG. 2 illustrates the detection of silencer activities by deletionanalysis. The precise end points of the deletion are indicated in basepairs from the AFP cap site. The closed bars indicate AFP 5'-flankingsequences and the dotted lines indicate deleted sequences. The CATactivities are expressed relative to pAF0.2(Bg)-CAT(pAF0.2) whichcontains the 169 bp AFP promoter region.

FIG. 3A illustrates the plasmids constructed to test the effect of theposition of the silencer region.

FIG. 3B illustrates the position dependent suppression of SV40 enhanceractivity by the Sd silencer region. This figure shows the CAT activitiesexpressed in HuH-7, huH-1/cl-2 and HeLa cells after transfection withCAT fusion genes shown in FIG. 3A. The smaller letters correspond to theplasmid constructs in FIG. 3A.

FIG. 4 illustrates the silencer region. The endpoints of deletion areindicated in base pairs from the AFP cap site. CAT activities expressedare shown as percentages of the pSV2-CAT activity above thecorresponding fragments. Hi, Hind III; Bg, BglII.

FIG. 5A illustrates the nucleotide sequence of the human AFP distalsilencer regions (SEQ ID NO:6). The 17 bp repeated elements areunderlined.

FIG. 5B illustrates the nucleotide sequence of the human AFP proximalsilencer region (SEQ ID NO:7). The 17 bp repeated element is underlined.

FIG. 5C is a comparison of the 17 bp repeated elements in the distal andproximal silencer regions.

FIG. 6A illustrates the CAT activity of constructs having the 31 bpsilencer region. C_(m), 1-Ac and 3-Ac are as defined in FIG. 1B.

FIG. 6B illustrates the nucleotide sequences of the wild-type (SEQ IDNO:8) and mutant (SEQ ID NO:9) fragments.

FIG. 6C illustrates the relationship between the number of copies of the31 bp Sd fragment and transcription suppressive activity.

FIG. 7 illustrates the structure of the diphtheria toxin gene andrestriction maps of pSV2-CAT, pSV2/HToxin (SEQ ID NOS:10 and 11), pTHX1and pTH1-176.

FIG. 8 indicates the CAT activities expressed in HuH-7 and HeLa cellstransfected with pSV2-CAT together with pSV2/H Toxin, pTH1-176 or pUC19.

FIG. 9 illustrates a restriction map of a pSV2/HT/neo^(r) plasmid intowhich a neomycin-resistant gene was incorporated and shows thetransformation frequency of G418 resistant colonies after transfectionwith various plasmids.

FIG. 10A illustrates restriction maps of: a DNA fragment containing theAFP gene and the transcriptional regulatory elements (a promoter P (SEQID NO:4 and 5), enhancers E_(A) (nucleotide regions from SEQ ID NO:12)and E^(B) (nucleotides 1700 to 1712 of SEQ ID NO:12), silencers S₁ (SEQID NO:6) and S₂ (SEQ ID NO:7) and a glucocorticoid reactive site G)located in the 5'-flanking region of the AFP gene; pAF4.9/HToxin; pAF4.9Δ2.7!/HToxin; pAF4.9 Δ2.7!(S)₈ /HToxin; pAF(AB)₂ (S)₈ /HToxin; pAF(AB)₁(S)₈ /HToxin; pAF(A)₂ (B)₁ (S)₈ /HToxin; pAF(A)₃ (B)₁ /HToxin;pAF0.2/HToxin; and pGEM/HToxin. The relative levels of CAT expressionfrom pSV2-CAT in HuH-7 and huH-1/cl-2 cells transfected with the aboveplasmids are indicated.

FIG. 10B illustrates the restriction maps of pSVAF(AB)₁ (S)₈ /HToxin andpSVAF(AB)₁ /HToxin. The relative levels of expression from pSV2-CAT inHuH-7 and huH-1/cl-2 cells transfected with the above plasmids areindicated.

FIG. 11A is a graph indicating the dose-dependency of the killing effectof various recombinant plasmids on HuH-7 cells.

FIG. 11B is a graph indicating the dose-dependency of the killing effectof various recombinant plasmids on huH-1/cl-2 cells.

FIG. 11C is a graph indicating the dose-dependency of the killing effectof various recombinant plasmids on HeLa cells.

FIG. 11D is a graph indicating the dose-dependency of the killing effectof various recombinant plasmids on COS7 cells.

FIG. 12 is the nucleotide sequence of 2.2 kb DNA between -5.1 and -2.9kb of the human AFP gene (SEQ ID NO:12). The 408 bp region used toconstruct pSVAF0.4-CAT is boxed. The solid arrow indicates an enhancercore sequence.

FIG. 13 illustrates the various mutant AFP promoters (PEII (SEQ IDNO:13) and MPEII (SEQ ID NO:14)). "72" indicates the 72 bp repeats fromthe SV₄₀ regulatory region.

FIG. 14 illustrates the relative CAT activity observed with variouswild-type and mutant AFP enhancers and wild-type and mutant silencers.

FIG. 15A illustrates the DNA (SEQ ID NO:26) and amino acid sequence (SEQID NO:27) of the Diphtheria Toxin A-chain.

FIG. 15B illustrates the DNA (SEQ ID NO:28) and amino acid sequence (SEQID NO:29) of the HToxin gene.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to a method for controlling the expressionof a heterologous gene in mammalian cells. The invention also relates toa method of destroying cancerous cells and a recombinant DNA constructfor use in such a method. Specifically, the present invention providesfor the combination of certain elements from the 5' regulatory region ofthe α-fetoprotein gene functionally linked to a heterologous gene forexpression in mammalian cells. In particular, it has been found that bycombining different silencer, enhancer and promoter elements, it ispossible to control the level of expression of a heterologous genefunctionally linked to the transcriptional regulatory region.

The albumin (ALB) and α-fetoprotein (AFP) genes exhibit extensivehomology with regard to nucleic acid sequence, gene structure, aminoacid sequence and protein secondary folding (for review see Ingram etal. PNAS 78 4694-4698 (1981). These genes are independently butreciprocally expressed in ontogeny. In normal development ALBtranscription steadily increases with fetal growth and continues at ahigh level throughout adulthood. Transcriptional expression of ALB inthe adult is confined to the liver. AFP is normally expressed in theyolk sac in fetal liver, and the fetal gastrointestinal tract, butdeclines after birth and is hardly detectable in non-pathologic ornon-regenerating adult liver or in other normal adult tissue. However,AFP transcription in adult liver often increases dramatically inhepatocellular carcinoma (HCC). In addition, transcription may also beelevated in non-seminomatous and fixed carcinoma of the testis; inendodermal sinus tumors, in certain teratocarcinomas, and in certaingastrointestinal tumors. Liver-specific expression of AFP and ALB is theresult of interactions of the regulatory seactivating, heir genes withtrans-activating, transcriptional factors found in nuclear extracts fromliver.

The methods of this invention may be used to control the expression ofheterologous genes in tissue culture. This may be important where highlevels of transcription of the heterologous genes are undesirable,because large concentrations of the heterologous protein within the cellleads to cell death. Further, where it is desired to express genes innormal liver cells, those genes may be linked to AFP regulatory regionswith the silencer region deleted to thereby allow the expression of thatheterologous gene in all liver cells. While other promoters may beuseful for this purpose, the AFP promoter and enhancer are preferredbecause the AFP promoter is a strong promoter with a high level oftranscriptional activity. Since the AFP regulatory elements areassembled in such a manner as to permit the expression of a linked genein liver, inborn errors of metabolism due to the lack or abnormality ofa gene may be corrected by the present invention. Such inborn errorsinclude phenylketonuria, urea cycle disorders, hemophiliacs, α₁-antitrypsin deficiency, and mucopolysaccharide deficiency. Furthermore,this method should be useful for treating viral hepatitis, such ascaused by hepatitis C virus, by introducing the gene encoding interferonwhich has been shown to be highly effective against HCV.

The methods of the present invention may also be useful in the treatmentof hepatocellular cancer. It has been shown by the inventors that ispossible to arrest the growth of, or kill, mammalian carcinoma cellswith a recombinant expression fragment comprising a AFP transcriptionalregulatory sequences linked to a toxin gene. This recombinant expressionvector may be incorporated into an infective virion. Upon administrationof an infective virion containing the recombinant expression fragment toa patient, the toxin is selectively expressed in the target cell. It hasbeen found that the level of expression of the toxin will vary dependingon the type of cancer cell. By the methods of the present invention, itis possible to adjust the level of expression of the toxin in the cells.The control of expression may be advantageous to prevent necrosis ofliver tissue in the patient or alternatively, to increase the rate. ofcell death for cancer cells which do not express large amount of AFPprotein.

In mammalian cells, certain genes are ubiquitously expressed. Mostgenes, however, are expressed in a temporal and/or tissue-specificmanner, or are activated in response to an extracellular inducer. Forexample, certain genes are actively transcribed only at very precisetimes in ontogeny in specific cell types or in response to some inducingstimulus. This regulation is mediated in part by the interaction betweentranscriptional regulatory sequences (which are for example promoter andenhancer regulatory DNA sequences), and sequence-specific, DNA-bindingtranscriptional protein factors.

In mammalian cells, normally two DNA sequences are required for thecomplete and efficient transcriptional regulation of genes that encodemessenger RNA's in mammalian cells: promoters and enhancers. "Promoters"are located immediately upstream (5') from the start site oftranscription. Promoter sequences are required for accurate andefficient initiation of transcription. A typical promoter includes anAT-rich region called a TATA box (which is located approximately 30 basepairs 5' from the start site of transcription initiation start site).

The activity of promoter sequences are modulated by other sequencescalled "enhancers". The "enhancer" sequence may be a great distance fromthe promoter in either an upstream, (5') or downstream (3') position.Hence, enhancers operate in an orientation- and position-independentmanner. However, based on similar structural organization and functionthat may be interchanged the absolute distinction between promoters andenhancers is somewhat arbitrary. Enhancers increase the rate oftranscription from the promoter sequence. It is predominantly theinteraction between sequence-specific transcriptional factors with thepromoter and enhancer sequences that enable mammalian cells to achievetissue-specific gene expression. The presence of these transcriptionalprotein factors (tissue-specific, trans-activating factors) bound to thepromoter and enhancers (cis-acting, regulatory sequences) enable othercomponents of the transcriptional machinery, including RNA polymerase,to initiate transcription with tissue-specific selectivity and accuracy.

The "silencer" is a DNA region which inhibits transcription initiationby interfering with enhancer activity.

The term "functionally linked" as used herein means that thetranscription regulatory region is linked to the heterologous gene insuch a manner that the expression of the heterologous gene is controlledby the transcription regulatory region.

The term "transcription regulatory region" as used herein includes allof the sequences involved in the transcription of a gene includingenhancers, silencers and promoters.

Similar to the regulatory structure of the ALB gene, the regulatoryelements of the AFP genes promote tissue-specific expression in certainliver pathologies, such as HCC (Mol. Cel. Biol. 6: 477-487 (1988);Science 235: 53-58 (1987). The regulatory elements of a mammalian AFPgene consist of a specific 5' promoter-proximal region (located in somemammalian species between 85 and 52 bp 5' to the gene). This sequence isessential for transcription in hepatomas. In addition, there areupstream (5') regulatory elements well defined for the murine AFP genewhich behave as classical enhancers (Mol. Cel. Biol. 6: 477-487 (1986);Science 235: 53-58 (1987). These upstream regulatory elements aredesignated elements I, II and III and are located between 1,000 to 7,600bp 5" to the transcription initiation site for the murine AFP gene.These three enhancer domains are not functionally equivalent atgenerating tissue-specific expression of AFP. Elements I and II have thegreatest capacity to direct liver-specific expression of AFP. It isimportant to note that the regulatory sequences of the AFP geneadvantageously contain the sequences not only for tissue-specifictranscriptional activation but also for repression of expression intissues which should not express AFP.

The regulatory regions of the human AFP gene have been characterized. Astructural gene placed in the correct orientation 3' to the wild-typeAFP regulatory sequences will enable that structural gene to beselectively expressed in fetal liver, hepatomas, non seminomatouscarcinomas of the testis, certain teratocarcinomas, certaingastrointestinal tumors and other normal and pathological tissues whichspecifically express AFP. The DNA fragment containing thetranscriptional regulatory region of the AFP gene, which is a part ofthe recombinant DNA of the present invention, should be a DNA fragmentobtained from the regulatory region of the AFP gene, that is, the5'-flanking region (the upstream region) of the AFP gene.

As used herein the term "AFP promoter" means a promoter from a mammalianAFP gene region. Preferably the promoter is from the human AFP generegion. More preferably the promoter comprises the sequence disclosed inFIG. 13 (SEQ ID NOS:4, 5, 13 and 14). The promoter may be the wild-typepromoter or it may be a mutated promoter. As used herein the term "AFPenhancer" means a DNA sequence encoding an enhancer from a mammalian AFPgene region. Preferably the enhancer is from the human AFP gene region.More preferably the enhancer comprises the sequences of domain A ordomain B as disclosed in FIG. 14 (SEQ ID NOS:15-24). The enhancer may bethe wild-type enhancer or a mutated enhancer. As used herein the term"AFP silencer" means a DNA sequence encoding a mammalian AFP silencer.Preferably the silencer is from the human AFP gene region. Morepreferably the silencer comprises the DNA sequence as disclosed in FIG.5 (SEQ ID NOS:6 and 7). The silencer may be the wild-type silencer or amutated silencer. The silencer may be the distal silencer or theproximal silencer.

The above-described transcriptional regulatory DNA fragment may beeither a native 5'-flanking sequence of the AFP gene or a recombinantDNA fragment containing one or more of the enhancer, silencer andpromoter of the AFP gene. The native 5'-flanking non-coding region ofthe AFP gene contains two enhancer sites, two silencer sites and onepromoter site, as shown in FIG. 10, whereas the recombinant DNA fragmentmay have variable numbers of enhancer and silencer elements.

Further, as shown in FIG. 10, the native AFP gene has a glucocorticoidresponsive element (g) in the transcriptional regulatory region. Underthe presence of glucocorticoid, the responsive element modifies theexpression of AFP. Therefore, when a recombinant DNA fragment containingthe glucocorticoid responsive element is used the expression of acancer-cell damaging protein can be regulated by administeringglucocorticoid.

The toxin gene may be a DNA fragment encoding a substance which alters,damages or kills not only cancer cells but also normal cells. Because,as described above, the expression of the gene in the recombinant DNAconstruct of the present invention is controlled by the transcriptionalregulatory region of the AFP gene, the DNA fragment encoding for such asubstance can be controlled such that it is transcribed only inAFP-producing cancer cells. Such genes may encode for direct toxins orindirect toxins. Examples of "direct toxins" are toxin genes, such asdiphtheria toxin (Maxwell, I. H., et al., Cancer Res., 46:4660-4664(1986)) and ricin Weiner, L. M., et al., 1989, Masui, H., et al., 1989!;cytokine genes Blankenstein, T., et al., J. Exp. Med., 173, 1047-1052(1991), Colombo, M. P., et al., J. Exp. Med., 173, 889-897 (1991),Leone, A., et al., Cell, 65, 25-35 (1991)!; tumor suppressor genes(Huang, M. J. S., et al., Science, 242, 1563-1566 (1988), Mercer, W. E.,et al., Proc. Natl. Acad. Sci. U.S.A., 88, 1958-1962 (1988)!; tumorvaccination genes, Wallich, R., et al., Nature, 315, 301-305 (1985),Rollins, B., et al., Mol. Cell. Biol., 11, 3125-3131 (1991)! DNAsequences that yield anti-sense RNA to oncogenes; genes encodingtumoroidal substances JE/McP-1; genes that produce anti-virussubstances; and genes that induce apoptosis.

Preferably, the direct toxin is diphtheria toxin (DT). This toxincatalyzes the transfer of the ADP-ribose moiety of NAD to elongationfactor 2 (EF-2) thereby blocking protein synthesis and causing celldeath. DT consists of 535 amino acids comprising two subunits designatedthe A and B chains (see FIG. 15A). The A chain (DT-A) which comprisesthe 193 amino-terminal residues catalyzes ADP-ribosylation of EF-2 andthe B chain (DT-B) which comprises the 342 carboxyl terminal residues(SEQ ID NO:27) promotes the binding of the toxin to cells and the entryof DT-A into the cytosolic compartment. DT-A is extremely toxic onceinside the cell and there is evidence that the introduction of a singlemolecule of this protein is lethal for a cell. (Yamaizumi et al. 1978)

More preferably, an attenuated form of the DT-A gene will be used inthis invention. For example, an attenuated form of DT-A (tox 176) hasbeen generated through chemical mutagenesis of the wild-type DT-A gene(SEQ ID NO:29) (Uchida et al. 1973; Yamaizumi et al. 1978) (see FIG.15B). The mutant toxin has been shown to be about 30-fold less toxicthan the wild-type protein. Such attenuated forms of toxin may enhancetarget cell specificity and prove to be highly versatile and effectiveagents for targeted cell killing.

Both the wild-type and mutant DT-A genes have been cloned and sequenced(Greenfield et al. (1983); PNAS 80: 6853-6857). They differ in thesecond position of codon 128, resulting in the substitution of asparticacid for a glycine in the deduced amino acid sequence. Several inactiveDT-A mutants have also been isolated (Giannini et al. 1984; Kaczorek etal., 1983). These studies have revealed amino acid residues critical forbiological functions of DT-A. Knowledge of the relationship betweensequence changes and diminished DT-A activity can be used by one skilledin the art to generate additional DT-A mutants with varying degrees ofcytotoxicity. One skilled in the art, given the disclosure of thisapplication, could generate recombinant DNA constructs for expression ofthe mutant toxin in cancer cells.

Alternatively an indirect toxin or enzyme pro-drug combinations may alsobe used; providing the enzyme is capable of selectively activating theadministered compound either directly or through an intermediate to acytostatic or cytotoxic metabolite. Equally the choice of compound willdepend on the enzyme system used, but must be selectively metabolized bythe enzyme either directly or indirectly to a cytotoxic or cytostaticmetabolite. One skilled in the art could select such a compound.

The varicella zoster virus (VZV) encodes a specific thymidine kinaseprotein. The gene has been cloned, sequenced and characterized (J. Gen.Virol. 67: 1759-1816)). The VZV thymidine kinase will, in contrast tothe mammalian enzyme, selectively monophosphorylate specific purinearabinosides and substituted pyrimidine compounds. 9-(β-D-arabinofuranosyl)-6methoxy-9H -purine is converted to9-β-D-arabinofuranosyl adenine tripohosphate Ara ATP! by this enzyme.European Patent Application No. 0 415 731 which is incorporated byreference herein!.

Other enzyme pro-drug combinations include the bacterial (for examplefrom Pseudomonas) enzyme carboxypeptidase G2 with the pro-drugpara-N-bis (2-chloroethyl) aminobenzoyl glutamic acid. Cleavage of theglutamic acid moiety from this compound releases a toxic benzoic acidmustard; alkaline phosphatase from, for example, calf intestine, willconvert inactive phosphorylated compounds such as etoposide-phosphate,doxorubicin-phosphate, mitomycin phosphate, to toxic dephosphorylatedmetabolites. Penicillin-V amidase will convert phenoxyacetamidederivatives of doxorubicin and melphalan to toxic metabolites and thefungal (for example from Fusarium oxysporum) cytosine deaminase willconvert 5-fluoroctosine to toxic 5-fluorouracil.

The heterologous gene for expression in mammalian cells may be any genecapable of being expressed in mammalian cells. For example, the gene maybe phenylalanine hydroxylase, urea cycle enzymes, clotting factors, α₁-antitrypsin, mucopolysaccharide or interferon.

The vector carrying such a recombinant DNA should be able to enter humancells and have no adverse effect on human normal cells. Examples of thevector are: plasmid vectors, retroviralvectors, and adenoviralvectors.

A variety of methods are available to introduce foreign genes intomammalian cells in vitro. The most popular techniques are the calciumphosphate precipitation method (Graham and van der Eb, 1973),DEAE-dextran-mediated transfection (Lopata et al. 1984), liposomemediated transfection (Cudd 1984) and electroporation (Potter et al.1984). In the calcium phosphate precipitation procedure, DNA iscoprecipitated with calcium phosphate to form insoluble particles whichare taken up by cells by phagocytosis.

The DEAE-dextran protocol is a highly efficient and reproducibletransfection procedure. A solution containing 2 mg/ml of the desiredDNA, 250 mg/ml of DEAE-dextran (Mν500.000; Sigma), 50 mM Tris-HCl,pH7.5, is filtered through 0.2μ filter and added to the culture medium.Following an incubation for 2 hours at 37° C., the medium is removed andthe cells washed three times with medium and cultured in growth mediumfor 58 hours.

Liposome--mediated transfection is another transfection procedure. Asolution containing 1 μg and 10 μl cationic liposomes (1 mg/ml) is addedto 0.5 ml serum-free medium. This is applied to cells and incubated at37° C. for 3 to 5 hours. The medium is then replaced with fresh growthmedium and cultured for 48 hours.

Electroporation uses an electric field to open up pores in the cellwhich allow entry of DNA molecules presumably through diffusion (Frommet al. 1985; Neumann et al. 1982; Wong and Neumann 1982).Electroporation is most conveniently done using suspension cultures,whereas the calcium phosphate precipitation method and theDEAE-dextran-mediated transfection are most easily done using monolayercultures.

The technique of retroviral infection of cells to integrate artificialgenes into mammalian cells in mammals employs retroviral shuttle vectorswhich are known in the art, (see for example Miller and Baltimore (1986)Mol. and Cell Biol. 6: 2895-2902). Essentially, retroviral shuttlevectors are generated using the DNA form of the retrovirus contained ina plasmid. These plasmids also contain sequences necessary for selectionand growth in bacteria. Retroviral shuttle vectors are constructed usingstandard molecular biology techniques well known in the art. Retroviralshuttle vectors have the parental endogenous retroviral genes (eg. gag,pol and env) removed and the DNA sequence of interest inserted, such asthe molecular chimaeras which have been described. They however, containappropriate retroviral regulatory sequences for viral encapsidation,proviral insertion into the target genome, message splicing, terminationand polyadenylation. Retroviral shuttle vectors can be derived from theMoloney murine leukemia virus (Mo-MLV) but it will be appreciated thatother retroviruses can be used such as the closely related Moloneymurine sarcoma virus. (European Patent Application No. 0 415 731 whichis incorporated herein by reference). Certain DNA viruses may also proveto be useful as a delivery system. The bovine papilloma virus BPV!replicates extrachromosomally so that delivery system based on BPV havethe advantage that the delivered gene is maintained in a nonintegratedmanner.

The advantages of a retroviral-mediated gene transfer system are thehigh efficiency of the gene delivery to the targeted tissue, sequencespecific integration regarding the viral genome (at the 5' and 3' longterminal repeat (LTR) sequences) and little rearrangements of deliveredDNA compared to other DNA delivery systems.

Accordingly in one embodiment of the present invention there is provideda retroviral shuttle vector comprising a DNA sequence comprising a 5'viral LTR sequence, a cis acting psi encapsidation sequence, arecombinant DNA construct and a 3' viral LTR sequence.

In one embodiment, and to help eliminate non-tissue-specific expressionof the molecular chimaera, the recombinant DNA construct may be placedin opposite transcriptional orientation to the 5' retroviral LTR. Inaddition, a dominant selectable marker gene may also be included whichis transcriptionally driven from the 5' LTR sequence. Such a dominantselectable marker gene may be the bacterial neomycin-resistance gene NEO(Aminoglycoside 3" phosphotransferase type II), which confers oneukaroytic cells resistance to the neomycin analogue G418 sulphate(GENETICIN) The NEO gene aids in the selection of packaging cells whichcontain these sequences. Other vectors containing a NEO gene as aselectable marker have been described, for example, the N2 vector(Science 230: 1395-1398 (1985).

A theoretical problem associated with retroviral shuttle vectors is thepotential of retroviral long terminal repeat (LTR) regulatory sequencestranscriptionally activating a cellular oncogene at the site ofintegration in the host genome. This problem may be diminished bycreating SIN vectors. SIN vectors are self-activating vectors whichcontain a deletion comprising the promoter and enhancer regions in theretroviral LTR. The LTR sequences of SIN vectors do nottranscriptionally activate 5' or 3' genomic sequences. Thetranscriptional inactivation of the viral LTR sequences diminishesinsertional activation of adjacent target cell DNA sequences and alsoaids in the selected expression of the delivered molecular chimaera. SINvectors are created by removal of approximately 299 bp in the 3' viralLTR sequence (Biotechniques 4 504-512 (1986).

Thus preferably the retroviral shuttle vector of the present inventionare SIN vectors.

Since the parental retroviral gag, pol and env genes have been removedfrom these shuttle vectors, a helper virus system may be utilized toprovide the gag, pol and env retroviral gene products necessary topackage or encapsidate the retroviral vector into an infective virion.This is accomplished by utilizing specialized "packaging" cell lines,which are capable of generating infectious, synthetic virus yet aredeficient in the ability to produce any detectable wild-type virus. Inthis way the artificial synthetic virus contains a chimaera of thepresent invention packaged into synthetic artificial infectious virionsfree of wild-type helper virus. This is based on the fact that thehelper virus that is stably integrated into the packaging cell containsthe viral structural genes, but is lacking the psi site, a cis actingregulatory sequence which must be contained in the viral genomic RNAmolecule for it to be encapsidated into an infectious viral particle.

In addition to removal of the psi site, additional alterations can bemade to the helper virus LTR regulatory sequences to insure that thehelper virus is not packaged in virions and is blocked at the level ofreverse transcription and viral integration.

Selectivity of expression may be additionally improved by selectiveinfection of liver cells. The retroviral env gene present in thepackaging cell line defines the specificity for host infection. The envgene used in constructing the packaging cell line is modified togenerate artificial, infective virions that selectively infecthepatocytes. As an example, a retroviral env gene introduced into thepackaging cell may be modified in such a way that the artificial,infective virion's envelope glycoprotein selectively infects hepatocytesvia the specific receptor mediated binding utilized by the hepatitis Bvirus (HBV).

HBV primarily infects hepatocytes via specific receptor mediatedbinding. The HBV proteins encoded by the pre-S1 and pre-S2 sequencesplay a major role in the attachment of HBV to hepatocytes (HepadnaViruses edited Robinson et al. 189-203, 205-221, 1987). The env gene ofthe packaging cell is modified to include the hepatocyte binding site ofthe large S HBCV envelope protein. Such modifications of the env geneintroduced into the packaging cell may be performed by standardmolecular biology techniques well known in the art and will facilitateviral uptake in the target tissue.

The infective virion according to the invention may be formulated bytechniques well known in the art and may be presented as a formulationwith a pharmaceutically acceptable carrier therefore. Pharmaceuticalacceptable carriers, in this instance, may comprise a liquid mediumsuitable for use as a vehicle to introduce the infective virion into thepatient. An example of such a carrier is saline. The infective virionmay be a solution or suspension in such a vehicle. Stabilizers andantioxidants and/or other excipients may also be present in suchpharmaceutical formulations which may be administered by intra-venous orintra-arterial infusion. In the case of treating HCC intra-hepaticarterial infusion may be advantageous.

The amounts and precise regime in treating a mammal, will of course bethe responsibility of the attendant physician, and will depend on anumber of factors including the type and severity of the condition to betreated. However, for HCC, an intrahepatic arterial infusion of theartificial infective virion at the titre of between 2×10⁵ and 2×10⁷colony forming units per ml (CFU/ml) infective virions is likely to besuitable for a typical tumor. Total amount of virions infused will bedependent on tumor size and would probably be given in divided doses.

Where the heterologous gene encodes for a metabolic enzyme, a metabolitemust also be administered to the mammal. The dose of the drug willadvantageously be in the range 0.1 to 250 mg per kilogram body weight ofrecipient per day, preferably 0.1 to 100 mg per kilogram body weight.One skilled in the art could determine the dosage to be administeredbased on a number of factors, including the metabolic enzymecontemplated.

The term "substantially complete kill" means that the number of viablecells has been reduced by a 1 log reduction, for example, from 10⁴ to10³ cells. More preferably, the number of viable cells has been reducedby 2 logs. Even more preferably, it has been reduced by 3 logs.

The following examples serve to illustrate the present invention butshould not be construed as a limitation thereof:

EXAMPLE 1

HuH-7 is a human hepatoma cell line producing AFP which was obtainedfrom Dr. J. Sato, Okayama University, Japan. (Nakabayashi et al., (1982)Cancer Res. 22:3858). HuH-7 was cultured in a chemically defined mediumISE-RPMI, which contains ethanolamine (30 μg/ml) in IS-RPMI. huH-1/cl-2is a clone isolated from the parental huH-1 human hepatoma cell line(Huh and Utakoji (1981) Gann 72:178-179). It was obtained from Dr. Huh,University of Tokyo, Japan and was cultured in ISE-RPMI with 1% fetalcalf serum. HeLa is a human cell line (Gey, (1952) Cancer Res. 12:264)which does not produce AFP. HeLa cells were cultured in ISE-RPMI with 5%fetal calf serum. COS 7 cells are an african green monkey kidney cellline which does not produce AFP. The COS 7 cells were obtained from ATCCand were cultured in ISE-RPMI with 5% fetal calf serum.

In all cases, transfection was performed by the calcium phosphateprecipitation method as described by Nakabayachi et al. (1989) J. Biol.Chem. 204: 266-271. In order to determine CAT activity, the transfectedcells were lysed by several cycles of freezing and thawing andcentrifuged at 15,000 rpm for 5 min. The supernatant was heated at 60°C. for 10 min. and analyzed for chloramphenicol acetyltransferase (CAT)activity according to Gorman et al. (1982) Mol. Cell. Biol. 2:1044. Thisheat treatment is essential for the detection of CAT activity inhuH-1/cl-2 cells. Similar heat treatment of extracts resulted in a 3.5fold increase in HuH-7 cell CAT activity.

pBR-CAT was constructed by inserting the HindIII-BamHI fragment obtainedfrom pSVO-CAT obtained from Dr. B. Howard, (NCI/NIH) (Gorman et al.(1982) Mol. Cell Biol. 2:1044) into pBR322 purchased from BRL,Gaitherburg Md. according to the method of Walker et al., (1983) Nature306:557. This plasmid contains the CAT coding sequence and the SV40polyadenlyation signal but lacks the SV40 enhancer and early promoterelements.

CAT fusion genes were constructed by linking the CAT gene to AFP5'-flanking sequences obtained by restriction enzyme digestion orpolymerase chain reaction and inserting them into the HindIII site ofpBR-CAT.

Specifically, pAF1.0-CAT was constructed by inserting the 980 bpsequence between -951 and +29 relative to the cap site of the human AFPgene into the HindIII site at the 5' end of the CAT gene of pBR-CAT.

To construct pAF5.1-CAT containing the 5.1 kb of the AFP 5'-flankingsequence, pAF1.0-CAT was digested completely with PstI, partially withEcoRI and a 5.7 kb DNA sequence containing a 0.9 kb EcoRI-HindIII humanAFP DNA fragment (-870 to +29), the CAT gene and the pBR322 replicationorigin was recovered. pHAL-2w-Eco4.2, (a clone containing 4.2 kb AFP DNAfrom -5.1 to -871 bp inserted at the EcoRI site of pBR322) was digestedcompletely with PstI and partially with EcoRI. A 4.9 kb DNA fragmentconsisting of the 4.2 kb AFP EcoRI DNA and the 748 bp PstI-EcoRI DNAfragment of pBR322 was isolated and ligated to the 5.7 kb DNA derivedfrom pAF1.0-CAT.

EXAMPLE 2

Expression of CAT gene driven by the AFP 5'-flanking DNA region in HuH-7and huH-1/cl-2 cells.

To determine whether the differences in AFP production in HuH-7 andhuH-1/cl-2 cells are due to differences in transcriptional regulatoryactivities of the AFP 5'-flanking regions, the cells were transfectedwith plasmids containing the CAT gene to which various lengths of AFP5'-flanking sequences were linked. Transfection and analysis of CATactivity were performed as described in Example 1. The results of thetest are shown in FIGS. 1A and 1B. The amounts of cell extract used andthe incubation times were 25 μg and 20 minutes for HuH-7 (lanes 1-5) and100 μg and 180 minutes for huH-1/cl-2 (lanes 6-10).

The 5.1 kb AFP 5'-flanking DNA which contains the full AFP enhancerregion (-4.9/-3.9 kb) supported a high level of CAT expression in HuH-7(FIG. 1B, lane 3) but only a low level of expression in huH-1/cl-2 (FIG.1B, lane 8). CAT expression support by the 1.0 kb AFP promoter regionwas also greater in HuH-7 than in huH-1/cl-2 (FIG. 1B, lanes 4 and 9).Thus there is a correlation between transcriptional activities of theAFP 5'-flanking sequences and the levels of AFP production in HuH-7 andhuH-1/cl-2, respectively.

Comparison of CAT activities supported by 5.1 kb (containing the AFPenhancer) and 1.0 kb (without the AFP enhancer) of the AFP 5'-flankingsequence showed that the AFP enhancer stimulated the AFP promoteractivity 25-fold in HuH-7 (FIG. 1B, lanes 3 and 4) but only 2-fold inhuH-1/cl-2 (FIG. 1B, lanes 8 and 9). These results indicate that inhuH-1/cl-2 cells the AFP enhancer does not stimulate the AFP promoter asmuch as in HuH-7 cells.

EXAMPLE 3

Analysis of AFP promoter and enhancer activities in HuH-7 and huH-1/cl-2cells

To show that enhancer activation of the AFP promoter is selectivelyinhibited in huH-1/cl-2 cells as suggested above, it is important toestablish that both the AFP enhancer and the promoter are active in thiscell line.

To analyze the functionality of the AFP promoter, the effect ofdexamethasone was tested on the AFP promoter activity. This test isbased on the observation that dexamethasone stimulates the AFP promoteronly when it is functional. (Nakabayashi et al., (1989) J. Biol. Chem.264:266) HuH-7 and huH-1/cl-2 cells were transfected with pAF1.0CATincubated with or without 3×10⁻⁶ M dexamethasone for 2 days and analyzedfor CAT activity. The amounts of extract and the incubation times were25 μg and 20 min. for HuH-7 cells (lanes 1 and 2) and 100 μg and 180minutes for huH-1/cl-2 cells (lanes 3 and 4). See FIG. 1C.

It was found that dexamethasone stimulated CAT activity supported by the1.0 kb AFP promoter 9-fold in huH-1/cl-2 and 12-fold in HuH-7 cells.This result indicates that the AFP promoter is functional in huH-1/cl-2cells.

Next the activity of the AFP enhancer was determined in HuH-7 andhuH-1/cl-2 cells. The 2.4 kb full enhancer region (-5.3/-2.9 kb) and twosubregions, 0.3 kb domain A (-4.0/-3.7 kb) and 0.4 kb domain B(-3.7/-3.3 kb) were individually tested in conjunction with SV40promoter.

To construct pSV1'-CAT, the plasmid pSV2-CAT was digested with AccI andSphI and the smaller fragment was removed. The remaining DNA was madeblunt-ended by treatment with the large fragment of DNA polymerase I andligated through Bg1II linkers. This plasmid contains only 30% of oneSV40 72 bp repeat sequence and the TATA box. pSVAF2.4-CAT wasconstructed by inserting the 2.4 kb BglII-BglII fragment between -5.3and -2.9 kb of the AFP gene prepared from λHAL-2W into the BglII site ofpSV1'-CAT. The plasmid λHAL-2W was described in Urano et al. (1984) Gene32:255-261.

pSVAF0.4-CAT was constructed by inserting the 408 bp HindII-HaeIIIfragment between -3.7 and -3.3 kb of the AFP gene which had beenblunt-ended into the BglII site of pSV1'-CAT. See FIG. 12.

pSVAF0.3-CAT was constructed by digesting PSVAF2.9-CAT with BglII. Thereleased 2.4-kb full enhancer fragment was digested with DraI andHindIII to release the 318-bp fragment. The HindIII end of this fragmentwas converted to blunt end, BglII liners were attached to both ends andthe fragment was inserted into the BglII site of pSV1'-CAT.

All of these enhancer regions stimulated the SV40 promoter activity toessentially the same degrees in HuH-7 and huH-1/cl-2 cells. See Table 1.

                  TABLE 1                                                         ______________________________________                                        Expression of CAT activity from Various AFT-CAT fusion genes.                 CAT activity                                                                  pSV2-CAT                                                                             pmol/h/        pSVAF- pSVAF- pSVAF- pSV1`-                                    μg of       2.4-CAT                                                                              0.3-CAT                                                                              0.4-CAT                                                                              CAT                                Cell line                                                                            protein %      %      %      %      %                                  ______________________________________                                        HuH-7  731     100    62  (44) 22  (16) 5.9 (4)  1.4 (1)                      huH-1/cl-2                                                                           46      100    68  (38) 25  (14) 7.2 (4)  1.8 (1)                      HeLa   32      100    3.5 (1.7)                                                                              3.4 (1.6)                                                                              3.2 (1.5)                                                                              2.1 (1)                      ______________________________________                                    

This finding indicates that the AFP enhancer elements are as active inhuH-1/cl1-2 cells as in HuH-7 cells. These results suggest that positivetranscription factors regulating the AFP enhancer and promoter are notlimiting in huH-1/cl-2 cells.

EXAMPLE 4

Negative control elements suppress AFP enhancer activity in huH-1/cl-2cells.

The examples described above supported the view that the AFP enhancer isunable to stimulate the promoter in huH-1.cl-2 cells. To test whetherthis is due to the action of negative regulatory elements locatedbetween the enhancer and the promoter, various lengths of DNA downstreamof the enhancer (at -2.9 kb) of the 5.1 kb AFP 5'-flanking sequence inpAF5. 1-CAT were deleted. (FIG. 2) To achieve the different deletions,the plasmid pAF5.1-CAT was digested completely with BglII and with SacIor BstNI or partially with HindIII. The SacI or BstNI or HindIII siteswere blunt ended with Klenow fragment and ligated to BglII linkers. Theplasmid was then religated. These deletions had little effect on CATexpression in HuH-7. In huH-1/cl-2, on the other hand, CAT expressionchanged with several deletions. In particular, significant increaseswere observed with deletions from -2.9 kb to -951 bp ( Δ2.0! in FIG. 2)(3.6 fold) and from -2.9 kb to -169 bp ( Δ2.7! in FIG. 2) (10-fold).These results suggest that at least two negative control regions existbetween -1822 and -169 bp; one from -1822 to -951 (distal silencer "Sd")and the other from -402 to -169 bp (proximal silencer "Sp"). Inaddition, a small but consistent increase in CAT activity was observedassociated with the deletion from -951 to -402 bp ( Δ2.5! in FIG. 2)which may suggest the presence of a third, weak negative control elementin this region.

The 875-bp Sd region was analyzed for transcription-suppressive activityby inserting it back into pAF5.1 Δ2.7!-CAT in which a 2.7 kb sequencefrom -2.9 to -169 bp had been deleted. The Sd DNA was cut from the AFPregion with HindIII and blunt ended with Klenow fragment. BglII linkerswere ligated to the 875-bp fragment and the fragment was inserted intothe BglII site in the plasmid pAF5.1 Δ2.7!-CAT. The resultant construct( Δ2.7!+875) showed a five-fold lower CAT activity than the parentalplasmid Δ2.7! in huH-1/cl-2 cells (FIG. 2). To further delimit thesilencer activity, the 875 bp DNA fragment was divided into twofragments by digestion with DraI enzyme to obtain a 409 bp 5'-fragment(from -1822 to -1414) and a 389 bp 3' fragment (from -1336 to -948), andeach fragment was tested separately for suppressive CAT activity. The409 bp 5' fragment ( Δ2.7!+409) suppressed CAT activity strongly,whereas the 3' fragment ( Δ2.7!+389) did so weakly (FIG. 2). Thisfinding indicates that the major silencer activity is contained in the409 bp 5' fragment. In the absence of the enhancer, the 5' fragmentweakly stimulated AFP promoter activity. These results indicate that theaction of Sd is to interfere with the AFP enhancer activity withoutaffecting AFP promoter activity.

In the absence of the entire suppressor region ( Δ2.7!), the CATactivity expressed in huH-1/cl1.2 is four fold lower than that in HuH-7(25.9 versus 116). This finding suggests that another mechanism existsin huH-1/cl-2 to suppress the intrinsic AFP promoter activity, althoughthis accounts only partially for the large difference in AFP productionbetween HuH-7 and huH-1/cl-2.

EXAMPLE 5

The Sd element suppresses SV40 enhancer activity in a position-dependentmanner

The effect of the Sd region on a heterologous enhancer was examined byinserting the 875 bp Sd region (from -1822 to -948 bp) into the BglIIsite in the SV40 enhancer of pSV1.6-CAT (SphI site converted to BglIIsite) in normal (d) and reverse orientations (e) (FIG. 3A). Theresultant constructs expressed much lower CAT activity in huH-1/cl-2cells than did the parental plasmid, indicating that Sd can suppress theheterologous SV40 enhancer in an orientation-independent manner.Suppression of CAT activity was also observed in HuH-7 and HeLa cellsalthough to much lesser extents. This finding suggests that the Sdactivity is not strictly cell type specific. To show that the observedeffects are not due to the disruption of the SV40 enhancer, a 924 bp DNA(from -4.9 to -4.0 kb) of the AFP gene with no known regulatory activitywas inserted at the BglII site of pSV1.6-CAT as a control (c) (FIG. 3B).No significant changes in CAT expression were observed with the 924 bpDNA insert as compared to pSV1.6-CAT.

The orientation-independent suppression was also observed with the 409bp Sd fragment in the SV40 enhancer. The 409 bp fragment (from -1822 to-1414 bp) was inserted into the AccI (a and b), BglII (f and g) or BamHI(h and i) site in normal and reverse orientations. When it was insertedupstream of the SV40 enhancer or downstream of the CAT gene, nosuppression effect was observed in either orientation in any of the celllines tested. These results show that the effect of Sd is orientationindependent but position dependent.

EXAMPLE 6

Identification of a silencer element

To delimit the Sd region that exerts transcriptional suppression, weinserted various lengths of Sd subfragments in the SV40 enhancer (BglIIsite) of pSV1.6-CAT (FIG. 4). The longest fragment is a HindIII fragmentof 875 bp. This fragment was blunt ended and BglII linkers were attachedand the fragment was inserted into the BglII site of the SV40 enhancer.In addition, two sub-fragments from the 875 bp HindIII fragment wereobtained by digesting the fragment with DraI and blunt ended. BglIIlinkers were attached and the fragments were inserted into the BglIIsite of the SV40 enhancer. The 31 bp fragment was synthesized by DNASynthesis Laboratory, University of Calgary as two single strands of 31bp and annealed these complementary strands to make double-stranded DNA.This was ligated into the BglII site of the SV40 enhancer.

Five fragments covering the region upstream of -1760 suppressed CATactivity. The shortest active fragment was 31 bp long, from -1790 to-1760. Four other fragments covering the region downstream of -1750 wereinactive (FIG. 4). The 229-bp Sp region from -402 to -174 was alsotested in a similar manner. It showed transcription-suppressiveactivity, although to a lesser degree than did Sd (FIG. 4).

We found that the 31-bp fragment which exhibited suppressive activitycontains a 17-bp stretch, 5'-CTTCATAACTAATACTT-3', which is repeatedfour times within a 90-bp region from -1810 to -1720 (FIG. 5A (SEQ IDNO:6), underlined). A similar sequence is also found in the Sp region(FIG. 5B (SEQ ID NO:7), underlined). In all cases, the first sixnucleotides, CTTCAT, and the last two, TT, are completely conserved(FIG. 5C). DNase I footprinting analysis by the method of Sawadaishi etal. (1988) Mol. Cell Biol. 8:5179-5187 showed that these sequences wereprotected by nuclear extracts prepared from huH-1/cl-2.

To further characterize the function of theses sequences, we insertedthe 31-bp (SEQ ID NO:8) oligonucleotide (from -1790 to -1760 bp) intothe BglII , AccI or BamHI site (SEQ ID NO:9) of pSV1.6-CAT. A mutantfragment with three-nucleotide substitutions was inserted into the BglIIsite. The mutant sequence was synthesized. These constructs weretransfected into huH-1/cl-2 or HuH-7 cells and 2 days later CATactivities were analyzed as described in Example 1. The amounts ofextract and incubation times were 25 μg and 20 min. for HuH-7 (lanes 1to 6) and 100 μg and 180 min. for huH-1/cl-2 (lanes 7 to 12). See FIG.6, lanes 1 and 7, pSV1.6-CAT (positive control); 2 and 8, wild-type 31bp fragment inserted in BglII ; 3 and 9, mutant fragment inserted inBglII ; 4 and 10, wild-type fragment inserted in AccI; 5 and 11,wild-type fragment inserted in BamHI; 6 and 12, pSV1'-CAT (negativecontrol).

The fragment inserted within the SV40 enhancer strongly suppressed CATactivity in huH-1/cl-2 (FIG. 6A, lane 8), but only a weak suppressionwas observed in HuH-7 (FIG. 6A, lane 2). The insertion of the fragmentupstream of the SV40 enhancer (FIG. 6A, lanes 4 and 10) or downstream ofthe CAT gene (FIG. 6A, lanes 5 and 11) had no effect on CAT activity ineither cell line. Substitution of three nucleotides within the 17-bprepeated sequence (SEQ ID NO:9) (FIG. 6B) resulted in loss ofsuppressive activity (FIG. 6A, lane 9). This finding confirms theassociation of the suppressive activity with this sequence.

To determine the relationship between transcriptional suppression andthe number of copies of the Sd elements, one, three, or eight copies ofthe 31-bp sequence (SEQ ID NO:8) was inserted into pAF5.1 Δ2.7!-CAT. The31 bp synthetic fragment which has BglII sites on both ends was ligatedinto the BglII site of pAF5.1 Δ2.7!-CAT. In order to obtain multiplecopies of the fragment, the 31 bp fragment was multimerized byself-ligation and then inserted into the BglII site of pAF5.1 Δ2.7!-CAT,which was then transfected into HuH-7 or huH-1/cl-2 cells. The level ofCAT activity was measured by the method of Example 1 (see FIG. 6C).Analysis of CAT expression from these constructs showed that CATactivity decreased with increasing number of copies of this sequence inhuH-1/cl-2 but not in HuH-7 (FIG. 6C). The CAT activities are expressedrelative to that of pAF0.2-CAT.

EXAMPLE 7

Referring to FIG. 7, the diphtheria toxin gene, a known gene, codes fordiphtheria toxin consisting of an A and B-chain. It is known thatdiphtheria toxin can kill eukaryotic cells by blocking proteinsynthesis. The B-chain is involved in the binding of the A-chain to acell. The sequence of the A and B chain of the DT gene is published.Greenfield, L., et al., Proc. Natl. Acad. Sci. U.S.A., 80, 6853-6857(1983)! See FIG. 15A (SEQ ID NOS:26 and 27). The DE-A gene and thetox-176 gene were obtained from Dr. Bernstein, Mount Sinai Hospital,Toronto. A DNA fragment coding for a modified A-chain (HToxin) (SEQ IDNOS:28 and 29) was used. This modified DNA fragment was made by usingPCR synthesis. This DNA fragment (HToxin) encodes for an A-chain havingCys as the 66th amino acid and Arg as the 194th amino acid, instead ofthe natural A-chain having Tyr and Pro, respectively, as reported byGreenfield and others. This DNA fragment was provided with a Hind IIIrestriction enzyme cleavage site at the N-terminal and a Hpalrestriction enzyme cleavage site at the C-terminal, using syntheticprimers containing the Hind III or the HpaI site, respectively.

pSV2-CAT obtained from Dr. B. H. Howard, NCI/NIH! Gorman, et al.. Mo.Cell. Biol. 2, 104 (1982)! was digested by Hind III and Hpal to removethe CAT gene, and the DNA fragment encoding the HToxin DNA was insertedinto the plasmid to construct a recombinant plasmid pSV2/HToxin. Thisrecombinant plasmid was used as a positive control because it isexpressed nonspecifically in most cell lines under the direction of theSV40 early promoter.

The plasmid pTHX1 was obtained from Dr. Bernstein, Mount Sinai Hospital,Toronto. It contains a wild-type DT-A gene next to the metallothioneingene promoter.

The plasmid pTHX-176 contains a mutant DT-A gene having Asn instead ofGly in the 128th position from the N-terminus. This plasmid was obtainedfrom Dr. A. Bernstein, Mount Sinai Hospital, Toronto.

The cell-killing effect of pSV2/HToxin was determined by comparing itwith the cell-killing effects of other plasmids. More specifically,HuH-7 cells were transfected by the method of Example 1 with pSV2-CATalone or with pTHX1, pTH1-176 or pSV2/HToxin. Then, the CAT activitiesexpressed by these transfected cells were determined. The ability of aparticular vector to suppress protein synthesis is indicated by thelevel of CAT activity. As the amount of protein synthesis is suppressed,the level of CAT activity decreases Nakabayashi H., et al., Mol. Cell.Biol., 11(12), 5885-5893 (1991)!. The results, shown in FIG. 8, indicatethat pTHX1 and pSV2/HToxin have substantially the same level ofsuppressing effect on the CAT activity and therefore substantially thesame level of cell-killing effect. pTH1-176 exhibited a level ofsuppressing effect about one third of the level exhibited by pTHX1.

The results indicated that the AFP regulatory sequences used directedthe expression of the HToxin gene in AFP producing hepatoma cells.

EXAMPLE 8

Effect of Diphtheria toxin on cell lines

The plasmid pSV2/HT/neo^(r) was obtained by linking the neomycinresistant gene (neo^(r)) Stratagene, La Jolla, Calif.! to theabove-mentioned pSV/HToxin. The plasmid pSV2/HToxin was idgested withBamHI and then with the Klenow fragment to form blunt ends. pMC1-neo^(r)(Stratagene, La Jolla Calif.) was treated with XhoI and SalI to releasethe neo^(r) gene (1083 bp). This was inserted into the treatedpSV2/HToxin plasmid above by blunt end ligation.

The plasmid pMC1-neo^(r) was obtained from Stratagene, La Jolla, Calif.

HuH-7 cells and HeLa cells were transfected with pSV2/HT/neo^(r) (seeFIG. 9) or a control plasmid pMC1-neo^(r) (Strategene) by the calciumphosphate method of Example 1. Transformants having neo^(r) wereselected by using G418 (GIBCO). Following transfection, cells weretreated with 50 μg/ml of G418 for two days and then with increasingamounts (up to 200 μg/ml for HuH-7 cells and 1 mg/ml for HeLa cells) for3-4 weeks. When the HeLa and HuH-7 cells were transfected with thecontrol plasmid, pMC1-neo, both cell lines formed colonies of thetransformants because the plasmid has the neo^(r) gene. On the otherhand, when the HeLa and HuH-7 cells were transfected with the plasmidpSV/HT/neo^(r) which has the neo^(r) gene, neither of the cell linesformed colonies because the plasmid pSV/HT/neo^(r) also has HToxin geneexpression, resulting in death of the transformants. Neither of the celllines transfected with pUC19 (BRL), which does not contain either of thegenes (i.e. a negative control) formed colonies (see Table 2). Theseresults prove that the HToxin gene has a cell-killing effect in bothHuH-7 and HeLa cells.

                  TABLE 2                                                         ______________________________________                                                     Number of Colonies (10.sup.5 cells)                              Plasmid        HuH-7      HeLa                                                ______________________________________                                        pMC1neo.sup.r  4.2        0.5                                                 pSV2/HToxin/neo.sup.r                                                                        0          0                                                   pUC19          0          0                                                   ______________________________________                                    

EXAMPLE 9

Effect of the Silencer Region on the Cell-killing by Diphtheria Toxin.

Various recombinant plasmids as shown in FIG. 10 were constructed bylinking the HToxin gene to DNA chains containing different lengths ofthe transcriptional regulatory regions of the AFP gene. Morespecifically, the above-mentioned pSV2/HToxin was digested by Hind IIIand BamHI to obtain a DNA fragment having 759 bp which included theHToxin gene. The 759 bp DNA fragment was inserted in between the HindIIIand BamHI sites of the pGEM-7Z vector (Promega), thus constructingpGEM7Z/HToxin. The plasmid pAF4.9/HToxin comprises the entiretranscriptional regulatory region of the AFP gene, and the HToxin genecontrolled by the transcriptional regulatory region.

The 4.9 kb 5'-flanking region of the AFP gene was obtained by partiallydigesting pAF5.1-CAT (Example 1) with HindIII, electrophoresing theresultant fragments in a 0.4% agarose, then collecting the 4.9 kb DNAfragment. The collected fragment was inserted into the HindIII site ofthe above-mentioned pGEM72/HToxin, thus obtaining the desired plasmid,pAF4.9/HToxin. pAF4.9 Δ2.7!/HToxin in which the 4.9 kb 5'-flankingregion of the AFP gene lacks a region from -2.9 kb to -169 bp, (i.e. thesilencer region) was obtained by partially digesting pAF5.1 Δ2.7!-CATwith HindIII and inserting the resultant 2.2 kb DNA fragment into theHind III site of pGEM/HToxin.

The plasmid pAF4.9 Δ2.7!(S)₈ /HToxin comprises the transcriptionalregulatory region of the AFP gene and the HToxin gene controlled by theAFP enhancer, 8 copies of the AFP silencer element and the promoter.This was obtained by partially digesting pAF5.1 Δ2.7!(S)₈ -CAT (Example6) with HindIII and inserting the 2.4 kb fragment having the enhancerand 8 copies of the silencer elements and the promoter into the HindIIIsite of pGEM/HToxin. pAF0.2/HToxin was obtained by the following steps:digestion of pAF4.9 Δ2.7!/HToxin by SmaI, ligation with a BglII linker,BglII digestion and removal of short fragments between the SmaI andBglII sites, and then BglII self-ligation. The plasmid pAF0.2/HToxin isa comparative example that lacks the enhancer and silencer sequences.

The plasmid pAF(AB)₂ (S)₈ /HToxin was generated by digesting pAF4.9Δ2.7!(S)₈ /HToxin with EcoRI and inserting the 72.8-bp DraI-HaeIIIfragment (-4.0/-3.3 kb) after attaching EcoRI linkers.

The plasmid pAF(AB)₁ (S)₈ /HToxin was generated by digesting pAF4.9Δ2.7!(S)₈ /HToxin with EcoRI and inserting two copies of the DraI-HaeIIIfragment (-4.0/-3.3 kb) after attaching EcoRI linkers. See FIG. 10B.

The plasmid pAF(A)₃ (B)₁ (S)₈ /HToxin was generated by digestingpAF(AB)₁ (S)₈ /HToxin with XbaI inserting two copies of DraI-HindIIIfragment (-4.0/-3.7 kb) after converting blunt ends and attaching XbaIlinkers.

The plasmid pAF (A)₃ (B)₁ /HToxin was generated by removing 8 copies ofsilencer element from pAF(A)₃ (B)₁ (S)₈ /HToxin by digestion with BglIIfollowed by religation.

Two hepatoma cell lines (HuH-7, huH-1) and two non-hepatic cell lines(HeLa, COS) were transfected with the recombinant plasmids containingthe HToxin gene together with pSV2-CAT. The cell-damaging effectsexpressed in the transfected cells were examined by determining thelevel of suppression of CAT expression caused by the HToxin expressionby the methods of Example 1. HuH-7 cells were cultured in ISE-RPMIalone, huH-1/cl-2 cells were cultured in ISE-RPMI with 1% fetal calfserum. The non-hepatic cell lines were cultured in ISE-RPMI with 5%fetal calf serum.

0.5×10⁵ cells/cm² of HeLa and COS7 Gluzman, Y., Cell, 23, 175-182(1981)! and 0.5×10⁵ cells/cm² of HuH-7 and huH-1/cl-2 Huh, N. andUtakoji, T., Gann, 72, 178-179 (1981)! were inoculated and transfectedwith 10 μg of pSV2/HToxin and various amounts of the above-describedrecombinant plasmids by the Ca²⁺ precipitation method Gorman et al.,Mol. Cell. Biol., 2, 104 (1982)!. After 48 hours, the cells werecollected and the CAT activities were determined. The results are shownin FIG. 11. As indicated in the figures, pSV2/HToxin strongly suppressedthe CAT activity in all the cell lines, and pAF4.9/HToxin stronglysuppressed the CAT activity in the AFP highly-producing hepatoma cellline HuH-7 (FIG. 1A) but did not suppress CAT activity in huH-1/cl-2FIG. 11B) and the non-hepatic cell lines, HeLa (FIG. 11C) or COS7(FIG.11D). pAF4.9 Δ2.7!/HToxin, lacking the silencer region, suppressed theCAT activity in HuH-7 and huH-1/cl-2. On the other hand, pAF4.9Δ2.7!(S)₈ /HToxin having 8 copies of silencer sequences suppressed theCAT activity in HuH-7 (FIG. 11A) but not in huH-1/cl-2 (FIG. 11b).pAF0.2/HToxin lacking the AFP transcriptional regulatory region exceptfor 200 bp of the promoter region did not suppress the CAT activity inany of the cell lines.

The results show that the HToxin gene can be expressed selectively inAFP highly-producing hepatoma cells by using recombinant DNA accordingto the present invention, such as pAF4.9/HToxin and pAF4.9 Δ2.7!(S)₈/HToxin.

The effect of increasing the number of AFP enhancers elements on theexpression of the CAT gene was determined to be as follows.

    ______________________________________                                                        Relative CAT Activity                                         ______________________________________                                        AFP enhancer domain A                                                                           1 copy   6.8                                                                  3 copies 52.4                                               AFP enhancer domain B                                                                           1 copy   3.3                                                                  3 copies 34.5                                               ______________________________________                                    

As described above, a recombinant DNA enters both normal and cancercells but causes the expression of the linked gene selectively inAFP-producing cells. Therefore, the recombinant DNA of the presentinvention can be suitably used as a gene cancer-therapeutic agent whichdamages or kills AFP-producing cells, e.g. hepatocellular carcinoma,without adversely affecting normal cells.

EXAMPLE 10

Generation of single-bose substitution mutations in the AFP promoter

To introduce single-base mutations in PE (proximal element) and MPE(most proximal element) sequences, an oligonucleotide-directed in vitromutagenesis system version 2 (Amersham) was used. This system was basedon the method of Eckstein and coworkers (Taylor et al., 1985a; Taylor etal. 1985b; Nakamaye and Eckstein, 1986; Sayers et al., 1988). Theprocedure utilized single-stranded pBS-200 plasmid DNA as a template.This single-stranded DNA was hybridized to an oligonucleotide primer(spanning PE or MPE regions) harboring a mutation at the desired site.The primed single-stranded pBS-200 plasmid was then extended to form thecomplementary strand. Subsequently, the wild type strand was removed,and resynthesized using the mutant strand as template. For this purpose,eight oligonucleotides were synthesized (supplied by The Regional DNASynthesis Laboratory, University of Calgary). To generate pointmutations in PE, the following oligonucleotides were used as mutantprimers: PE mutant I, PE mutant II, PE mutant III, PE mutant IV (seeTable 3).

To generate point mutations in MPE, the following oligonucleotides wereused as mutant primers: MPE mutant I, MPE mutant II, MPE mutant III, andMPE mutant IV (see Table 3). The mutated pBS-200 PE and MPE! plasmidswere introduced into E. coli XL-1Blue host bacteria. In order to obtainthe correct mutations, pBS-200 plasmids prepared from 12 to 48 bacterialcolonies were screened by single-lane sequencing. The correct mutantswere further confirmed by sequencing the entire sequences.

                  TABLE 3                                                         ______________________________________                                        List of oligonucleotide primers used in this study                                     SEQ                                                                           ID                                                                   Names    NO:    Sequences (5` to 3`)                                          ______________________________________                                        CAT      30     dCAACGGTGGTATATCCAGTG                                         AdML     31     dAGTCATGCCCGCTTTTGAGA                                         Υ-globin                                                                       32     dGTGATAGTAGCCTTGTCCTC                                         PE mutant I                                                                            33     dATGCTGTGAATTATTG                                             PE mutant II                                                                           34     dCTGTTAAGTATTGGCA                                             PE mutant III                                                                          35     dTTAATTATGGGCAAAT                                             PE mutant IV                                                                           36     dATTATTGGAAAATGTC                                             MPE mutant I                                                                           37     dCAAAAGGTGACTAGTT                                             MPE mutant II                                                                          38     dAGGTTACGAGTTAACA                                             MPE mutant III                                                                         39     dTTACTAGTGAACAGGC                                             MPE mutant                                                                             40     dCTAGTTAAAAGGCATT                                             IV                                                                            Pr-169 BglII                                                                           41     dGATCAGATCTTACAAATAACCGCTATGC                                                 TG                                                            PR-98 BglII                                                                            42     dGATCAGATCTTTTCAACCTAAGGAAATA                                                 CC                                                            KS       43     dCGAGGTCGACGGTATCG                                            SK       44     dTCTAGAACTAGTGGATC                                            T3       45     dATTAACCCTCACTAAAG                                            t7       46     dTAATACGACTCACTATAGGG                                         ______________________________________                                    

This mutagenesis created plasmid PEII, MPEII and PEII, MPEII which areshown in FIG. 13 (SEQ ID NOS:4, 5, 13 and 14). The plasmids weretransfected into HuH-7 cells by the methods of Example 1.

The level of promoter activity was measured by measuring the CATactivity by the methods of Example 1. The results show that allmutations resulted in decreased promoter activity, particularly MPEII.

EXAMPLE 11

Generation of single-dose substitution mutations in the AFP enhancer andsilencer regions

Wild-type and mutant enhancer and silencer elements were synthesized(FIG. 14, SEQ ID NOS:8, 15 to 25) and inserted into the BglII site ofpAF0.2-CAT. The resulting plasmids were transfected into HuH-7 cells bythe methods of Example 1. The level of CAT activity was determined bythe method of Example 1. The results indicate that the wild-typeenhancer elements stimulated CAT expression in a highly dose-dependentmanner, i.e., 3 copies of enhancer elements resulted in about a 10-foldincrease in CAT expression over one copy of enhancer element. The mutantenhancer, on the other hand, showed much less stimulatory activity anddose dependency.

While the present invention has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

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    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 46                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GTCAATTAGTAAC13                                                               (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GTTAGTAATTACT13                                                               (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GTTAATAATCTAC13                                                               (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GTTAATTATTGGC13                                                               (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GTTACTAGTTAAC13                                                               (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 409 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       AGCTTATATAGTTTGCTTCATAAAACTCTATTTCAGTTCTTCATAACTAATACTTCATGA60                CTATTGCTTTTCAGGTATTCCTTCATAACAAATACTTTGGCTTTCATATATTTGAGTAAA120               GTCCCCCTTGAGGAAGAGTAGAAGAACTGCACTTTGTAAATACTATCCTGGAATCCAAAC180               GGATAGACAAGGATGGTGCTACCTCTTTCTGGAGAGTACGTGAGCAAGGCCTGTTTTGTT240               AACATGTTCCTTAGGAGACAAAACTTAGGAGAGACACGCATAGCAGAAAATGGACAAAAA300               CTAACAAATGAATGGGAATTGTACTTGATTAGCATTGAAGACCTTGTTTATACTATGATA360               AATGTTTGTATTTGCTGGAAGTGCTACTGACGGTAAACCCTTTTTGTTT409                          (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 234 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       TGGCATATGATAGGCATTTAATAGTTTTAAAGAATTAATGTATTTAGATGAATTGCATAC60                CAAATCTGCTGTCTTTTCTTTATGGCTTCATTAACTTAATTTGAGAGAAATTAATTATTC120               TGCAACTTAGGGACAAGTCATCTCTTTGAATATTCTGTAGTTTGAGGAGAATATTTGTTA180               TATTTGCAAAATAAAATAAGTTTGCAAGTTTTTTTTTTCTGCCCCAAAGAGCTC234                     (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GATCTCAGTTCTTCATAACTAATACTTCATGACTAGATC39                                     (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GATCTCAGTTGTTCGTAAGTAATACTTCATGACTAGATC39                                     (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      AAGCTTAAGCATATGGGCGCC21                                                       (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      TGATAATAGTTATC14                                                              (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2240 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GAATTCTTAGAAATATGGGGGTAGGGGTGGTGGTGGTAATTCTGTTTTCACCCCATAGGT60                GAGATAAGCATTGGGTTAAATGTGCTTTCACACACACATCACATTTCATAAGAATTAAGG120               AACAGACTATGGGCTGGAGGACTTTGAGGATGTCTGTCTCATAACACTTGGGTTGTATCT180               GTTCTATGGGGCTTGTTTTAAGCTTGGCAACTTGCAACAGGGTTCACTGACTTTCTCCCC240               AAGCCCAAGGTACTGTCCTCTTTTCATATCTGTTTTGGGGCCTCTGGGGCTTGAATATCT300               GAGAAAATATAAACATTTCAATAATGTTCTGTGGTGAGATGAGTATGAGAGATGTGTCAT360               TCATTTGTATCAATGAATGAATGAGGACAATTAGTGTATAAATCCTTAGTACAACAATCT420               GAGGGTAGGGGTGGTACTATTCAATTTCTATTTATAAAGATACTTATTTCTATTTATTTA480               TGCTTGTGACAAATGTTTTGTTCGGGACCACAGGAATCACAAAGATGAGTCTTTGAATTT540               AAGAAGTTAATGGTCCAGGAATAATTACATAGCTTACAAATGACTATGATATACCATCAA600               ACAAGAGGTTCCATGAGAAAATAATCTGAAAGGTTTAATAAGTTGTCAAAGGTGAGAGGG660               CTCTTCTCTAGCTAGAGACTAATCAGAAATACATTCAGGGATAATTATTTGAATAGACCT720               TAAGGGTTGGGTACATTTTGTTCAAGCATTGATGGAGAAGGAGAGTGAATATTTGAAAAC780               ATTTTCAACTAACCAACCACCCAATCCAACAAACAAAAAATGAAAAGAATCTCAGAAACA840               GTGAGATAAGAGAAGGAATTTTCTCACAACCCACACGTATAGCTCAACTGCTCTGAAGAA900               GTATATATCTAATATTTAACACTAACATCATGCTAATAATGATAATAATTACTGTCATTT960               TTTAATGTCTATAAGTACCAGGCATTTAGAAGATATTATTCCATTTATATATCAAAATAA1020              ACTTGAGGGGATAGATCATTTTCATGATATATGAGAAAAATTAAAAACAGATTGAATTAT1080              TTGCCTGTCATACAGCTAATAATTGACCATAAGACAATTAGATTTAAATTAGTTTTGAAT1140              CTTTCTAATACCAAAGTTCAGTTTACTGTTCCATGTTGCTTCTGAGTGGCTTCACAGACT1200              TATGAAAAAGTAAACGGAATCAGAATTACATCAATGCAAAAGCATTGCTGTGAACTCTGT1260              ACTTAGGACTAAACTTTGAGCAATAACACACATAGATTGAGGATTGTTTGCTGTTAGCAT1320              ACAAACTCTGGTTCAAAGCTCCTCTTTATTGCTTGTCTTGGAAAATTTGCTGTTCTTCAT1380              GGTTTCTCTTTTCACTGCTATCTATTTTTCTCAACCACTCACATGGCTACAATAACTGTC1440              TGCAAGCTTATGATTCCCAAATATCTATCTCTAGCCTCAATCTTGTTCCAGAAGATAAAA1500              AGTAGTATTCAAATGCACATCAACGTCTCCACTTGGAGGGCTTAAAGACGTTTCAACATA1560              CAAACCGGGGAGTTTTGCCTGGAATGTTTCCTAAAATGTGTCCTGTAGCACATAGGGTCC1620              TCTTGTTCCTTAAAATCTAATTACTTTTAGCCCAGTGCTCATCCCACCTATGGGGAGATG1680              AGAGTGAAAAGGGAGCCTGATTAATAATTACACTAAGTCAATAGGCATAGAGCCAGGACT1740              GTTTGGGTAAACTGGTCACTTTATCTTAAACTAAATATATCCAAAACTGAACATGTACTT1800              AGTTACTAAGTCTTTGACTTTATCTCATTCATACCACTCAGCTTTATCCAGGCCACTTAT1860              TTGACAGTATTATTGCGAAAACTTCCTAACTGGTCTCCTTATCATAGTCTTATCCCCTTT1920              TGAAACAAAAGAGACAGTTTCAAAATACAAATATGATTTTTATTAGCTCCCTTTTGTTGT1980              CTATAATAGTCCCAGAAGGAGTTATAAACTCCATTTAAAAAGTCTTTGAGATGTGGCCCT2040              TGCCAACTTTGCCAGGAATTCCCAATATCTAGTATTTTCTACTATTAAACTTTGTGCCTC2100              TTCAAAACTGCATTTTCTCTCATTCCCTAAGTGTGCATTGTTTTCCCTTACCGGTTGGTT2160              TTTCCACCACCTTTTACATTTTCCTGGAACACTATACCCTCCCTCTTCATTTGGCCCACC2220              TCTAATTTTCTTTCAGATCT2240                                                      (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GTTAAGTATTGGC13                                                               (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      GTTACGAGTTAAC13                                                               (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      AATTCTTTCTAATACCAAAGTTCAGTTTACTGTTCCAGATC41                                   (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      AATTCTTTCTAATACCGACGCTCCGTTTACTGTTCCAGATC41                                   (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      GATCAGAATTACATCAATGCAAAGATC27                                                 (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      GATCACAATTACGTCAACGCAAAGATC27                                                 (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      GATCTTGTTTGCTGTTAGCATACAAACTCGATC33                                           (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      GATCTAGCTTGCTGTTAGCATACCAGCTCGATC33                                           (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      GATCTATTTTTCTCAACCACTCACATGGCTACAAGATC38                                      (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      GATCTATTTTTCTCAACTACCCACATGGCTACAAGATC38                                      (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      GATCCTGATTAATAATTACACTAAGTCAAGATC33                                           (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      GATCCTGATTAATAAGTACACTAAGTCAAGATC33                                           (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      GATCTCAGTTGTTCGTAACTAAGACTTCATGACTAGATC39                                     (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 582 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..582                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      GCAGGCGCTGATGATGTTGTTGATTCTTCTAAATCTTTTGTGATGGAA48                            AlaGlyAlaAspAspValValAspSerSerLysSerPheValMetGlu                              151015                                                                        AACTTTTCTTCGTACCACGGGACTAAACCTGGTTATGTAGATTCCATT96                            AsnPheSerSerTyrHisGlyThrLysProGlyTyrValAspSerIle                              202530                                                                        CAAAAAGGTATACAAAAGCCAAAATCTGGTACACAAGGAAATTATGAC144                           GlnLysGlyIleGlnLysProLysSerGlyThrGlnGlyAsnTyrAsp                              354045                                                                        GATGATTGGAAAGGGTTTTATAGTACCGACAATAAATACGACGCTGCG192                           AspAspTrpLysGlyPheTyrSerThrAspAsnLysTyrAspAlaAla                              505560                                                                        GGATACTCTGTAGATAATGAAAACCCGCTCTCTGGAAAAGCTGGAGGC240                           GlyTyrSerValAspAsnGluAsnProLeuSerGlyLysAlaGlyGly                              65707580                                                                      GTGGTCAAAGTGACGTATCCAGGACTGACGAAGGTTCTCGCACTAAAA288                           ValValLysValThrTyrProGlyLeuThrLysValLeuAlaLeuLys                              859095                                                                        GTGGATAATGCCGAAACTATTAAGAAAGAGTTAGGTTTAAGTCTCACT336                           ValAspAsnAlaGluThrIleLysLysGluLeuGlyLeuSerLeuThr                              100105110                                                                     GAACCGTTGATGGAGCAAGTCGGAACGGAAGAGTTTATCAAAAGGTTC384                           GluProLeuMetGluGlnValGlyThrGluGluPheIleLysArgPhe                              115120125                                                                     GGTGATGGTGCTTCGCGTGTAGTGCTCAGCCTTCCCTTCGCTGAGGGG432                           GlyAspGlyAlaSerArgValValLeuSerLeuProPheAlaGluGly                              130135140                                                                     AGTTCTAGCGTTGAATATATTAATAACTGGGAACAGGCGAAAGCGTTA480                           SerSerSerValGluTyrIleAsnAsnTrpGluGlnAlaLysAlaLeu                              145150155160                                                                  AGCGTAGAACTTGAGATTAATTTTGAAACCCGTGGAAAACGTGGCCAA528                           SerValGluLeuGluIleAsnPheGluThrArgGlyLysArgGlyGln                              165170175                                                                     GATGCGATGTATGAGTATATGGCTCAAGCCTGTGCAGGAAATCGTGTC576                           AspAlaMetTyrGluTyrMetAlaGlnAlaCysAlaGlyAsnArgVal                              180185190                                                                     AGGCGA582                                                                     ArgArg                                                                        (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 194 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      AlaGlyAlaAspAspValValAspSerSerLysSerPheValMetGlu                              151015                                                                        AsnPheSerSerTyrHisGlyThrLysProGlyTyrValAspSerIle                              202530                                                                        GlnLysGlyIleGlnLysProLysSerGlyThrGlnGlyAsnTyrAsp                              354045                                                                        AspAspTrpLysGlyPheTyrSerThrAspAsnLysTyrAspAlaAla                              505560                                                                        GlyTyrSerValAspAsnGluAsnProLeuSerGlyLysAlaGlyGly                              65707580                                                                      ValValLysValThrTyrProGlyLeuThrLysValLeuAlaLeuLys                              859095                                                                        ValAspAsnAlaGluThrIleLysLysGluLeuGlyLeuSerLeuThr                              100105110                                                                     GluProLeuMetGluGlnValGlyThrGluGluPheIleLysArgPhe                              115120125                                                                     GlyAspGlyAlaSerArgValValLeuSerLeuProPheAlaGluGly                              130135140                                                                     SerSerSerValGluTyrIleAsnAsnTrpGluGlnAlaLysAlaLeu                              145150155160                                                                  SerValGluLeuGluIleAsnPheGluThrArgGlyLysArgGlyGln                              165170175                                                                     AspAlaMetTyrGluTyrMetAlaGlnAlaCysAlaGlyAsnArgVal                              180185190                                                                     ArgArg                                                                        (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 585 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..585                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      ATGGGCGCCGATGATGTTGTTGATTCTTCTAAATCTTTTGTGATGGAA48                            MetGlyAlaAspAspValValAspSerSerLysSerPheValMetGlu                              151015                                                                        AACTTTTCTTCGTACCACGGGACTAAACCTGGTTATGTAGATTCCATT96                            AsnPheSerSerTyrHisGlyThrLysProGlyTyrValAspSerIle                              202530                                                                        CAAAAAGGTATACAAAAGCCAAAATCTGGTACACAAGGAAATTATGAC144                           GlnLysGlyIleGlnLysProLysSerGlyThrGlnGlyAsnTyrAsp                              354045                                                                        GATGATTGGAAAGGGTTTTATAGTACCGACAATAAATACGACGCTGCG192                           AspAspTrpLysGlyPheTyrSerThrAspAsnLysTyrAspAlaAla                              505560                                                                        GGATGCTCTGTAGATAATGAAAACCCGCTCTCTGGAAAAGCTGGAGGC240                           GlyCysSerValAspAsnGluAsnProLeuSerGlyLysAlaGlyGly                              65707580                                                                      GTGGTCAAAGTGACGTATCCAGGGCTGACGAAGGTTCTCGCACTAAAA288                           ValValLysValThrTyrProGlyLeuThrLysValLeuAlaLeuLys                              859095                                                                        GTGGATAATGCCGAAACTATTAAGAAAGAGTTAGGTTTAAGTCTCACT336                           ValAspAsnAlaGluThrIleLysLysGluLeuGlyLeuSerLeuThr                              100105110                                                                     GAACCGTTGATGGAGCAAGTCGGAACGGAAGAGTTTATCAAAAGGTTC384                           GluProLeuMetGluGlnValGlyThrGluGluPheIleLysArgPhe                              115120125                                                                     GGTGATGGTGCTTCGCGTGTAGTGCTCAGCCTTCCCTTCGCTGAGGGG432                           GlyAspGlyAlaSerArgValValLeuSerLeuProPheAlaGluGly                              130135140                                                                     AGTTCTAGCGTTGAATATATTAATAACTGGGAACAGGCGAAAGCGTTA480                           SerSerSerValGluTyrIleAsnAsnTrpGluGlnAlaLysAlaLeu                              145150155160                                                                  AGCGTAGAACTTGAGATTAATTTTGAAACCCGTGGAAAACGTGGCCAA528                           SerValGluLeuGluIleAsnPheGluThrArgGlyLysArgGlyGln                              165170175                                                                     GATGCGATGTATGAGTATATGGCGCAAGCCTGCGCAGGTAACCGTGTC576                           AspAlaMetTyrGluTyrMetAlaGlnAlaCysAlaGlyAsnArgVal                              180185190                                                                     AGGCCATGA585                                                                  ArgPro                                                                        195                                                                           (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 194 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      MetGlyAlaAspAspValValAspSerSerLysSerPheValMetGlu                              151015                                                                        AsnPheSerSerTyrHisGlyThrLysProGlyTyrValAspSerIle                              202530                                                                        GlnLysGlyIleGlnLysProLysSerGlyThrGlnGlyAsnTyrAsp                              354045                                                                        AspAspTrpLysGlyPheTyrSerThrAspAsnLysTyrAspAlaAla                              505560                                                                        GlyCysSerValAspAsnGluAsnProLeuSerGlyLysAlaGlyGly                              65707580                                                                      ValValLysValThrTyrProGlyLeuThrLysValLeuAlaLeuLys                              859095                                                                        ValAspAsnAlaGluThrIleLysLysGluLeuGlyLeuSerLeuThr                              100105110                                                                     GluProLeuMetGluGlnValGlyThrGluGluPheIleLysArgPhe                              115120125                                                                     GlyAspGlyAlaSerArgValValLeuSerLeuProPheAlaGluGly                              130135140                                                                     SerSerSerValGluTyrIleAsnAsnTrpGluGlnAlaLysAlaLeu                              145150155160                                                                  SerValGluLeuGluIleAsnPheGluThrArgGlyLysArgGlyGln                              165170175                                                                     AspAlaMetTyrGluTyrMetAlaGlnAlaCysAlaGlyAsnArgVal                              180185190                                                                     ArgPro                                                                        (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      CAACGGTGGTATATCCAGTG20                                                        (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      AGTCATGCCCGCTTTTGAGA20                                                        (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      GTGATAGTAGCCTTGTCCTC20                                                        (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      ATGCTGTGAATTATTG16                                                            (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      CTGTTAAGTATTGGCA16                                                            (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      TTAATTATGGGCAAAT16                                                            (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      ATTATTGGAAAATGTC16                                                            (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      CAAAAGGTGACTAGTT16                                                            (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      AGGTTACGAGTTAACA16                                                            (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      TTACTAGTGAACAGGC16                                                            (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      CTAGTTAAAAGGCATT16                                                            (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      GATCAGATCTTACAAATAACCGCTATGCTG30                                              (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      GATCAGATCTTTTCAACCTAAGGAAATACC30                                              (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      CGAGGTCGACGGTATCG17                                                           (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      TCTAGAACTAGTGGATC17                                                           (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      ATTAACCCTCACTAAAG17                                                           (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      TAATACGACTCACTATAGGG20                                                        __________________________________________________________________________

What is claimed is:
 1. An isolated silencer region comprising thesilencer sd mutant sequence in FIG. 14 (SEQ ID NO:25).
 2. A recombinantexpression vector comprising the silencer region according to claim 1.3. A cell comprising the recombinant expression vector according toclaim
 2. 4. An isolated nucleic acid sequence comprising at least sixcopies of a human AFP silencer region and at least one copy of apromoter sequence functionally linked to a direct toxin gene.
 5. Arecombinant expression vector comprising the nucleic acid sequenceaccording to claim
 4. 6. A cell comprising the recombinant expressionvector according to claim
 5. 7. The nucleic acid sequence according toclaim 4 wherein the human AFP silencer region is selected from the groupconsisting of nucleotides 16-32 of SEQ ID NO:6; nucleotides 33-62 of SEQID NO:6; nucleotides 53-69 of SEQ ID NO:6; nucleotides 81-97 of SEQ IDNO:6 and nucleotides 86-102 of SEQ ID NO:7 or their complements.
 8. Thenucleic acid sequence according to claim 4 wherein the human AFPsilencer region comprises the sequence CTTCATAACTAATACTT (nucleotides38-55 OF SEQ ID NO:6) or its complement.
 9. The nucleic acid sequenceaccording to claim 4 wherein the human AFP silencer region comprises thesequence of FIG. 5A (SEQ ID NO:6) or its complement.
 10. The nucleicacid sequence according to claim 4 wherein the human AFP silencer regioncomprises the sequence of FIG. 5B (SEQ ID NO:7) or its complement. 11.The nucleic acid sequence according to claim 4 wherein the direct toxingene is selected from the group consisting of diphtheria toxin,diphtheria toxin chain A, ricin cytokine genes, tumor suppressor genesand tumor vaccination genes.
 12. The nucleic acid sequence according toclaim 4 wherein the direct toxin gene is diphtheria toxin chain A.